Much of the metabolic reprogramming seen in cancer cells, known as the Warburg effect, is regulated by the MYC oncoprotein. Among MYC-regulated changes are: addiction to exogenous glutamine and increased transcription of the lactate dehydrogenase-A gene. MYC also regulates expression of the cad gene, which encodes three enzymes in the pyrimidine biosynthesis pathway, as well as other enzymes linked to nucleotide biosynthesis. Another MYC-regulated change is increased mitochondrial biogenesis and mitochondrial genome (mtDNA) content, despite a decrease in ATP generation via the mitochondrial electron transport chain. Via a screen designed to isolate MYC targets whose transcription is tightly correlated with malignant transformation, the mitochondrial RNA polymerase (POLRMT) gene was identified by our group. POLRMT levels are significantly elevated in many human cancers, including various forms of leukemia, lymphoma, myeloma, melanoma, and epithelial tumors. POLRMT is a nuclear gene that encodes the single-chain polypeptide RNA polymerase responsible for all transcription in the mitochondria. The McMahon group has demonstrated that MYC controls transcription of the mitochondrial genome via POLRMT. Genetic rescue experiments show that induction of POLRMT is both necessary and sufficient for MYC to cause increased mtDNA content. Remarkably, blocking induction of POLRMT by MYC causes a synthetic lethality whereby the cells undergo apoptosis. MYC plays a critical role in glutamine/pyrimidine metabolism (glutamine is a precursor for pyrmidine biosynthesis), which may be explained by MYC's control of mtDNA replication. The dependence of pyrimidine biosynthesis on mtDNA content is based on one of the critical enzymes in the pathway residing within the inner mitochondrial membrane and requiring the ETC for activity. Decreased ETC function, elicited by loss of mtDNA content, inhibits the essential pyrimidine pathway enzyme dihydooratate dehydrogenase (DHOD). Thus, POLRMT induction is critical for pyrimidine biosynthesis. Coupled with MYC's tight control of glutamine metabolism and cad transcription, these observations suggest pyrimidine metabolism as a critical effector pathway for MYC. In this proposal, I will define the precise requirement for POLRMT in protection from MYC-induced apoptosis. More specifically, I will define the biochemical/metabolic event(s) that POLRMT controls to confer survival on MYC expressing cells and define the role of mtDNA loss in apoptosis. Additionally, I explore POLRMT as a possible therapeutic target for treating cancer.